Probable person to person transmission of novel avian influenza A (H7N9) virus in Eastern China, 2013: epidemiological investigation

BMJ 2013; 347 doi: http://dx.doi.org/10.1136/bmj.f4752 (Published 6 August 2013)
Cite this as: BMJ 2013;347:f4752

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Dear Editor,

A recent paper by Qi and colleagues in this journal described a detailed investigation of influenza A(H7N9) virus infections in two members from the same household (1). Based on the epidemiological investigation and reportedly high sequence similarity, the authors concluded that this provided the first evidence for probable human-to-human transmission. After reading the investigation of this A(H7N9) family cluster we would appreciate if Qi and colleagues could comment on the molecular findings described below, as based on these we reach a different conclusion.

We compared the influenza A(H7N9) sequences from the cluster by maximum parsimony analysis. Because maximum parsimony analysis does not assume a specific distribution and is best suited for analyzing sequences that are quite similar, this simple approach is the method of choice for analyzing defined virus outbreaks(2). The character data from a concatenated full-length HA, NA, and PB2 gene nucleotide alignment were used to build a maximum parsimony network in BioNumerics software using the Fitch method with a greedy tree construction algorithm, followed by random branch swapping to find the optimal network topology. We previously used this type of analysis to reconstruct a transmission network of highly pathogenic avian influenza A(H7N7) viruses during an outbreak that struck the Netherlands in 2003, resulting in infection of at least 89 humans, including three secondary cases (3-5). The obtained transmission network demonstrated a high level of agreement with epidemiologic data. Sequence analysis of A(H7N7) viruses obtained from human cases was performed on all available clinical samples and virus isolates and demonstrated absence of virus adaptation to MDCK-I cells within the regions sequenced. Moreover, the combined full length HA, NA and PB2 sequences corresponding with an A(H7N7) virus obtained from a culler could subsequently be identified in the A(H7N7) positive samples obtained from his daughter (100% identical) and wife (1 synonymous substitution, 2 days later). This A(H7N7) family cluster, characterized by virtually identical HA, NA and PB2 sequences, occurred within the three months period of the Dutch A(H7N7) virus outbreak where the maximum genetic distance of concatenated HA, NA and PB2 segments of A(H7N7) viruses was 25 nucleotide substitutions.

Since the recent identification of A(H7N9) in China, 135 laboratory confirmed human cases have been reported, including 44 fatal cases (WHO; 12 August 2013) (6, 7). Significant A(H7N9) sequence diversity has been observed, reflecting ongoing cycles of transmission (8). We postulated that a confirmed cluster of cases would have identical or highly similar virus sequences (9). However, when the viruses obtained from the father (A/Wuxi/2/2013), the daughter (A/Wuxi/1/2013) and from the live poultry market (A/environment/Wuxi/1/2013) together with other A(H7N9) outbreak virus sequences available from GenBank were used to build a maximum parsimony network, we came to a different conclusion (Figure 1.). All three A(H7N9) sequences from viruses obtained from the family cluster are located at different branches in the network, with large (≥12) nucleotide distances between them that reflect the observed genetic distances in the entire A(H7N9) outbreak over time and space.

The large nucleotide distances between individual A(H7N9) virus sequences are suggestive for surveillance gaps. For the A(H7N7) outbreak in the Netherlands, approximately 30% of the farm and human derived A(H7N7) HA, NA and PB2 sequences were identical to their presumed source of infection while ~70% of the A(H7N7) sequence displayed on average <2 nucleotide changes compared with their source of infection. To explain the large (≥12) nucleotide distances between the A(H7N9) family cluster strains, prolonged virus infection might have played a role. The index was sampled more than three weeks after disease onset, and the daughter was sampled ten days after disease onset. It is, however, highly unlikely that the large nucleotide distances between A(H7N9) viruses in the family cluster is explained by virus isolation using cell culture, given comparative studies of A(H7N7) sequences from clinical samples and culture isolates, and the number of passages required for positive selection to occur (10). Instead, the high sequence diversity within the A(H7N9) family cluster compared with total A(H7N9) outbreak sequence diversity, combined with the observation that the A(H7N9) family cluster sequences all are located on different branches of the transmission network, we would conclude that this almost certainly shows absence of human-to-human transmission within the reported A(H7N9) family cluster.

References:

1. Qi X, Qian YH, Bao CJ, Guo XL, Cui LB, Tang FY, et al. Probable person to person transmission of novel avian influenza A (H7N9) virus in Eastern China, 2013: epidemiological investigation. BMJ. 2013;347:f4752. Epub 2013/08/08.
2. Kolaczkowski B, Thornton JW. Performance of maximum parsimony and likelihood phylogenetics when evolution is heterogeneous. Nature. 2004;431(7011):980-4.
3. Jonges M, Bataille A, Enserink R, Meijer A, Fouchier RA, Stegeman A, et al. Comparative analysis of avian influenza virus diversity in poultry and humans during a highly pathogenic avian influenza A (H7N7) virus outbreak. J Virol. 2011;85(20):10598-604. Epub 2011/08/19.
4. Koopmans M, Wilbrink B, Conyn M, Natrop G, van der Nat H, Vennema H, et al. Transmission of H7N7 avian influenza A virus to human beings during a large outbreak in commercial poultry farms in the Netherlands. Lancet. 2004;363(9409):587-93.
5. Fouchier RA, Schneeberger PM, Rozendaal FW, Broekman JM, Kemink SA, Munster V, et al. Avian influenza A virus (H7N7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome. Proceedings of the National Academy of Sciences of the United States of America. 2004;101(5):1356-61.
6. Gao R, Cao B, Hu Y, Feng Z, Wang D, Hu W, et al. Human Infection with a Novel Avian-Origin Influenza A (H7N9) Virus. N Engl J Med. 2013. Epub 2013/04/13.
7. Kageyama T FS, Takashita E, Xu H, Yamada S, Uchida Y, Neumann G, Saito T, Kawaoka Y, Tashiro M. . Genetic analysis of novel avian A(H7N9) influenza viruses isolated from patients in China, February to April 2013. . Euro Surveilance. 2013;18(15):pii=20453.
8. Jonges M, Meijer A, Fouchier R, Koch G, Li J, Pan J, et al. Guiding outbreak management by the use of influenza A(H7Nx) virus sequence analysis. Euro Surveill. 2013;18(16). Epub 2013/04/25.
9. Jonges M, Rahamat-Langendoen J, Meijer A, Niesters HG, Koopmans M. Sequence-based identification and characterization of nosocomial influenza A(H1N1)pdm09 virus infections. The Journal of hospital infection. 2012;82(3):187-93. Epub 2012/09/28.
10. Hoper D, Kalthoff D, Hoffmann B, Beer M. Highly pathogenic avian influenza virus subtype H5N1 escaping neutralization: more than HA variation. J Virol. 2012;86(3):1394-404. Epub 2011/11/18.

Figure legend:

Figure 1.) Maximum parsimony analysis using A(H7N9) sequence data of concatenated HA, NA and PB2 sequences obtained from the family cluster and Genbank.

Competing interests: None declared

Marcel Jonges, virologist

Adam Meijer, Marion Koopmans

Department of Virology, Centre for Infectious Disease Control, National Institute for Public Health and the Environment, P.O. Box 1, 3720 BA, Bilthoven, The Netherlands

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All of the three scenarios regarding the possible transmission of the second case that Dr. Leung described are theoretically correct. However, as we mentioned in our paper, the most likely source of infection of the second case (the daughter) was from the index case (her father).

The third scenario was very less likely on the basis of the high similarity of all 8 genes from the two strains isolated from the father and the daughter.

Regarding the second scenario, its occurrence was also less likely, although that cannot be completely ruled out. Previous studies have demonstrated that poultry from live-bird markets was the important source of infection of human H7N9 cases (1-3). We did not interview the second case herself due to the fact that she was criticaly ill. However, from her family members, including her mother and husband, we knew clearly that her daily duty was to take care of her 22 month old daughter and she had never visited a live-bird market before the onset of her illness, which was very different from her father, who used to visit the nearby two live-bird markets before the onset of his illness and bought six quails one day between March 1 and March 4. Thus, the possibility of both the two cases obtaining their infections from a common source from the live-bird market was comparative low.

Was there another possibility that they became infected from their living environment (common household and residential district)? Field investigation discovered that no poultry was found in their living environment except two black swans that had been raised by an employee of the property management of the residential district for about two years. No positive results using rRT-PCR were detected from the cloacal swab and faecal samples collected from the two black swans. These data indicate that the two cases were unlikely to have become infected from their living environment. Moreover, the supposed incubation period of the second case would be 17 to 21 days based on the hypothesis that she got the infection in the same way as her father, which was much longer than that expected (4).

In conclusion, through our field epidemiological and laboratory investigation, we believe that the second case (the daughter) got the infection directly from the index case (her father), although the second scenario (both the cases got infections from a common source) cannot be completely ruled out.

Dr. Liu mentioned in his rapid response that one person cannot kill six quails alone. That is not correct. The index patient purchased the quails and had them killed by a special butcher just in the market where this service was provided. Aside from this, he did not buy any other live poultry 10 days before the onset of his illness, as we found through interviewing his wife and his son in law on multiple occasions. And no other live poultry was found in their living environment except for the two black swans. As for the potential social crisis resulting from our conclusion, we think that this is a little exaggerated and sensationalist claim. We just discovered an existing phenomenon from an aspect of science and research.

1. Bao CJ, Cui LB, Zhou MH, Hong L, Gao GF, Wang H. Live-Animal Markets and Influenza A (H7N9) Virus Infection. N Engl J Med. 2013 May 22.
2. Chen Y, Liang W, Yang S, Wu N, Gao H, Sheng J, et al. Human infections with the emerging avian influenza A H7N9 virus from wet market poultry: clinical analysis and characterisation of viral genome. The Lancet. 2013.
3. Shi J, Deng G, Liu P, Zhou J, Guan L, Li W, et al. Isolation and characterization of H7N9 viruses from live poultry markets — implication of the source of current H7N9 infection in humans. Chinese Science Bulletin. 2013:1-7.
4. Cowling BJ, Jin L, Lau EH, Liao Q, Wu P, Jiang H, et al. Comparative epidemiology of human infections with avian influenza A H7N9 and H5N1 viruses in China: a population-based study of laboratory-confirmed cases. Lancet. Jul 13;382(9887):129-37.

Competing interests: None declared

Wang Hua, epidemiologist

Ming-hao Zhou, Chang-jun Bao, Lun-biao Cui, et al.

Jiangsu Provincial Center for Disease Control and Prevention, No. 172, Jiangsu Rd. Nanjing

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Dear Editor,

The A(H7N9) influenza virus [1] and the MERS CoV [2] are currently attracting much attention because of the possibility of their global spread. Qi et al report the case of a probable person-person transmission of the A(H7N9) virus in Eastern China.[3] No sustained human-human transmission of the virus has been observed. From an epidemiological perspective, this is associated with a basic reproductive number less than 1 and, therefore, with no epidemic spread. However, because of the possibility of virus mutation and increased virus adaptation to humans, and the possibility of super-spreaders and favourable epidemic pathways, global efforts to combat the entry and development of the viruses in human populations continue with the early identification of cases and source identification at the forefront of these efforts.

Complex network epidemiology [4] provides a framework for addressing virus control measures. In particular, scale-free networks incorporate the realism of hubs and the possibility of super-spreaders in epidemic modelling. The stochastic modelling of epidemics enables us to understand how different epidemic sizes can arise for the same value of the basic reproductive number and how extreme epidemic events can arise.[5] In a recent study [6] of the dynamics of a virus spreading stochastically by contact in a scale-free network defined on a real-life human population distribution, the effect of various control measures were investigated. In particular, it was found that a reduction of viral transmission probability at the hubs of the network decreased the occurrence of the larger-sized (extreme) epidemic events. Public health measures should thus include the proactive identification of potential epidemic hubs in human societies and preemptive hub epidemic control measures. However, for a really effective epidemic control, an early reduction in transmission at and in the vicinity of the index case is essential. This requires reinforced public health surveillance systems for the early identification of index cases and of sources. Both hub transmission control and reduction at source need to be implemented to prevent the global spread of the A(H7N9) influenza virus and the MERS CoV.

References
1. Zhang Q, Shi J, Deng G et al. H7N9 Influenza Viruses Are Transmissible in Ferrets by Respiratory Droplet. Science. 2013;341(6144):410-4.
2. Breban R, Riou J, Fontanet A. Interhuman transmissibility of Middle East respiratory syndrome coronavirus: estimation of pandemic risk. Lancet 2013.
3. Qi X, Qian YH, Bao CJ, et al. Probable person to person transmission of novel avian influenza A (H7N9) virus in Eastern China, 2013: epidemiological investigation. BMJ2013;347:f4752
4. Colizza V, Barthélemy M, Barrat A et al. Epidemic modeling in complex realities. C. R. Biologies 2007;330:364–374.
5. Watts DJ, Muhamad R, Medina DC et al. Multiscale, resurgent epidemics in a hierarchical metapopulation model. Proc Natl Acad Sci. 2005;102(32):11157-62.
6. Moheeput K, Goorah SSD, Ramchurn SK. Spreading dynamics of a viral Infection in a complex network. WASET 2013;79:453-457.

Competing interests: None declared

Satish K Ramchurn, Associate Professor, Department of Physics

Smita Goorah, Senior Lecturer, Department of Medicine

University of Mauritius, Faculty of Science, Reduit, Mauritius

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To the Editor:

Xian qi et al[1] make an important contribution to the discussion about the probable person to person transmission of novel avian influenza A (H7N9) virus in Eastern China. However, their epidemiological investigation does not effectively support their conclusion. As noted by Xian Qi et al, the index patient purchased six quails in one of the live markets and cooked for the family one day between 1 March and 4 March. In Chinese life style, the fact that one person alone killed six quails for meal is unlikely, which also needs help of other family members. However, even the exact date was not remembered. During the past days, the index patient may buy some other live birds for home cooking, which they have forgotten now.

Secondly, a famliy live in the residential district, which was about 1 million square metres nearby live poultry markets. The special living environment created many opportunities for live poultry to humans, many of these were so tiny that we cannot perceive them.

Finally, even “the probable person to person transmission of novel avian influenza A (H7N9) virus” is a shocking conclusion, which may induce serious social crisis. Please look before you leap unless you have solid evidence for publishing such paper.

Yueju Liu, MD
Third hospital of Hebei Medical University
Number 139,Ziqiang Road,Shijiazhuang City,Hebei China liuyueju1983@gmail.com

References
1. Xian Qi, Yan-Hua Qian,Chang-Jun Bao,et al.Probable person to person transmission of novel avian influenza A (H7N9) virus in Eastern China, 2013: epidemiological investigation.BMJ2013;347:f4752

Competing interests: None declared

Yueju Liu, Orthopedic Surgeon

Third hospital of Hebei Medical University, Number 139 Ziqiang Road

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Since late February 2013, more than 130 laboratory-confirmed cases of human infections by the novel avian influenza A(H7N9) have been reported in China [1]. Although the number of cases has been declining since April, the latest confirmed case reported in Guangdong [2] shows that A(H7N9) still merits close attention. Whereas the severity of A(H7N9) disease appears higher than for seasonal influenza viruses [3], transmissibility between humans has been low to date with a small number of family clusters [4], and among 2,554 traced close contacts of confirmed H7N9 cases, only 4 (<0.2%) potential secondary infections were detected [5].

Qi et al. [6] reported an investigation into possible human-to-human transmission from a 60-year old father to his 32-year old daughter (the Jiangsu family cluster reported in [4]). By stating that they believe their investigation supports ‘probable’ human-to-human transmission, the authors give the impression that they believe there is more than a 50% chance that the daughter was infected by the father. The authors have conceded that there is an absence of certainty over the chain of transmission that led to infection of the daughter [6]. There is certainly a ‘possibility’ (i.e. a probability between 0 and 1) that (1) the daughter was infected directly by the father, but there is also a possibility that (2) the daughter and father were both infected from a common source, and furthermore a possibility that (3) the daughter and father were infected from separate sources. The third scenario is very unlikely given the close genetic relationship of the viruses isolated from the father and the daughter [6]. However, the authors did not demonstrate that scenario (1) had higher probability than scenario (2), or that scenario (1) had a probability above 50%.

There is support for scenario (1) from the epidemiologic investigation, where the daughter had no known poultry exposure and had prolonged close contact with the father while he was ill. In addition, sequencing of all eight genes from the three H7N9 strains isolated separately from the father and daughter demonstrated a high level of sequence similarity (99.6 – 99.9%). However, evidence against scenario (1) includes the relatively long apparent incubation period, if infection occurred during the period of greatest unprotected exposure. Moreover,while the viral genomes are very similar, this does not in itself imply transmission: we have previously reported 99.99% average sequence identity between human influenza viruses in index and secondary cases in households [7] and the rate with which sequence variation arises within infection and is transmitted to subsequent hosts is unknown. Additional mutations could have occurred during passaging in cell culture, and it would have been preferable if possible to sequence the virus directly from the original clinical specimens. The significance of the sequence similarity between the virus isolated from the father and daughter is unclear without further information and notably expanded sampling, as such observations may readily arise via alternative transmission scenarios. The single environmental isolate provides inadequate context, and as such, the genomic data do not add a great deal to the evidence for direct transmission.

This work highlights the need for a formal quantitative framework for the analysis of epidemiological and virus sequence data, allowing investigators to test formal hypotheses such as comparing the probability of scenario (1) vs (2) described above. Such a framework could be particularly valuable when assessing any future changes in transmissibility of H7N9.

*References*

1. Number of confirmed human cases of avian influenza A(H7N9) reported to WHO: Report 8 - data in WHO/HQ as of 30 May 2013, 15:45 GMT+1. Secondary Number of confirmed human cases of avian influenza A(H7N9) reported to WHO: Report 8 - data in WHO/HQ as of 30 May 2013, 15:45 GMT+1 2013. http://www.who.int/influenza/human_animal_interface/influenza_h7n9/08_Re....
2. Human infection with avian influenza A(H7N9) virus – update 11 August 2013 Secondary Human infection with avian influenza A(H7N9) virus – update 11 August 2013 2013. http://www.who.int/csr/don/don_updates/en/index.html.
3. Yu H, Cowling BJ, Feng L, et al. Human infection with avian influenza A H7N9 virus: an assessment of clinical severity. Lancet 2013;382(9887):138-45.
4. Li Q, Zhou L, Zhou M, et al. Preliminary Report: Epidemiology of the Avian Influenza A (H7N9) Outbreak in China. N Engl J Med 2013.
5. Cowling BJ, Jin L, Lau EH, et al. Comparative epidemiology of human infections with avian influenza A H7N9 and H5N1 viruses in China: a population-based study of laboratory-confirmed cases. Lancet 2013;382(9887):129-37.
6. Qi X, Qian YH, Bao CJ, et al. Probable person to person transmission of novel avian influenza A (H7N9) virus in Eastern China, 2013: epidemiological investigation. BMJ 2013;347:f4752.
7. Poon LL, Chan KH, Chu DK, et al. Viral genetic sequence variations in pandemic H1N1/2009 and seasonal H3N2 influenza viruses within an individual, a household and a community. J Clin Virol 2011;52(2):146-50.

Competing interests: BJC has received research funding from MedImmune Inc. and consults for Crucell NV.

Nancy H. L. Leung, PhD student

Colin J. Worby, William P. Hanage, Marc Lipsitch, Benjamin J. Cowling

Division of Epidemiology and Biostatistics, School of Public Health, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, China

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Dear Editor,

Qi et al reported first probable case of human-to-human transmission of novel avian influenza A (H7N9) virus[1]. However, symptom-guided laboratory investigations of contacts suggest that H7N9 has yet to acquire an ability to maintain sustained human-to-human transmission, even though serological examination has not been performed among asymptomatic contacts to exclude silent transmission[2,3]. At the current zoonotic stage of the H7N9 infection, it would be logical to target control measures close to the poultry sources. However, there has been conspicuous absence of H7 and / or N9 serology data among involved poultry and occupational exposed workers in markets and farms with documented outbreaks[2,3]. In sharp contrast with H5N1, H7N9 infection among poultry has been largely silent, posing a genuine problem for effective surveillance and control[2-4]. Virological yield from poultry using reverse transcriptase-polymerase chain reaction was very low even at putative outbreak sources[4]. With the short turnover of poultry in markets or even farms, serological evidence of exposure among occupational exposed workers could be more informative at this stage to inform control strategy at the animal sources, especially in view of the very low H7 or N9 titres in stored blood samples taken from poultry workers in 2012 in Eastern China[5].

To avert a possible devastating pandemic of the H7N9, timely research efforts are desperately needed to inform public health actions. There is perhaps little room for complacency.

Chi Chiu LEUNG*, David SC HUIϯ, Paul KS CHANǂ
*Tuberculosis and Chest Service, Department of Health, Hong Kong, China
ϯDepartment of Medicine & Therapeutics, Faculty of Medicine, The Chinese University of Hong Kong
ǂDepartment of Microbiology, Faculty of Medicine, The Chinese University of Hong Kong

Corresponding Author: Dr. Chi Chiu Leung
Wanchai Chest Clinic, 99 Kennedy Road, Wanchai, Hong Kong, China
Email: cc_leung@dh.gov.hk

References

1. Qi X, Qian YH, Bao CJ, et al. Probable person to person transmission of novel avian influenza A (H7N9) virus in Eastern China, 2013: epidemiological investigation. BMJ2013;347:f4752
2. Cowling BJ, Jin L, Lau EH, et al. Comparative epidemiology of human infections with avian influenza A H7N9 and H5N1 viruses in China: a population-based study of laboratory-confirmed cases. Lancet. 2013;382:129-137.
3. Gao HN, Lu HZ, Cao B, et al. Clinical findings in 111 cases of influenza A (H7N9) virus infection. N Engl J Med. 2013;368:2277-85.
4. Shi JZ, Deng GH, Liu PH, et al. Isolation and characterization of H7N9 viruses from live poultry markets—Implication of the source of current H7N9 infection in humans. Chin Sci Bull, doi: 10.1007/s11434-013-5873-4.
5. Bai T, Zhou J, Shu Y. Serologic study for influenza A (H7N9) among high-risk groups in China. N Engl J Med. 2013;368:2339-40.

Competing interests: None declared

Chi C Leung, Consultant Chest Physician

David SC HUI, Paul KS CHAN

Tuberculosis and Chest service, 1/F, Wanchai Chest Clinic, 99 Kennedy Road, Wanchai, Hong Kong

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Dear Editor,

I read this paper with great interest, but have a remark on Table 1. The table is a bit confusing as for each gene segment in the upper half above the 100% similarity diagonal (indicated with a dash) the percent similarity is given whereas in the lower half below the dashed diagonal the percent difference is given. The current layout of the table suggests all numbers are percent identity (similarity). Did the authors mean with "divergence" in the Gene segment column heading actually the percent divergence for the lower half of data for each gene segment? Just providing the percent identity (similarity) would have been enough to underpin that the father and daughter sequences were slightly more alike each other than alike the environmental virus.

Competing interests: None declared

Adam Meijer, virologist

National Institute for Public Health and the Environment, PO Box 1, 3720 BA, Bilthoven, the Netherlands

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